Animal Cell Culture: Definition, Media, Growth, Techniques, Applications & Scale-Up

Table of Content

Introduction to Tissue Culture
History of Animal Cell Culture
Eukaryotic Cells in Culture
Types of Animal Tissue Culture
Contact Inhibition
Behavior of Normal and Cancer Cells in Culture
Cell Morphology Categories
Classification of Animal Cell Culture
Methods of Obtaining Cell Culture
Cell Culture Systems
Factors Affecting Subculturing Frequency
Subculturing
Cell Lines and Their Types
Characteristics of Immortal and Cancer Cells
Commonly Used Cell Lines
Physical Environment for Culturing Animal Cells
Culture Media
Components of Basal Media
Osmolarity and Temperature of Culture Medium
Types of Culture Media
Major Equipment Used in Animal Cell Culture Laboratory
Cryopreservation of Cell Culture
Cell Counting
Cell Viability
Confluency
Applications of Animal Cell Culture Technology
Scale-Up of Animal Cell Culture

Introduction to Tissue Culture

Tissue culture is a general term used for the removal of cells, tissues, or organs from an animal or plant and their placement into an artificial environment that supports their survival and growth.
The artificial environment used in tissue culture usually consists of a suitable glass or plastic culture vessel that contains a liquid or semisolid culture medium.
This culture medium provides all the essential nutrients required for the survival, growth, and maintenance of the cultured cells, tissues, or organs.
Tissue culture has been known since the beginning of the twentieth century as an important method for studying the behavior, growth, and characteristics of animal cells under controlled laboratory conditions.

History of Animal Cell Culture

The history of animal cell culture began in 1856 when Ludwig was able to keep animal organs alive outside the body by continuously pumping blood through them.
In 1907, Ross Harrison demonstrated the in vitro growth of living animal tissue by explanting a fragment of nerve cord from a frog tadpole and placing it in a drop of frog lymph, and he is considered by many to be the father of cell culture.
Between 1911 and 1912, Burrows and Alexis Carrel successfully grew tissue fragments from adult dogs, cats, rats, and guinea pigs, and they also managed to culture malignant (cancerous) tissues.
Alexis Carrel initiated a cell line from the heart tissue of a chick embryo, which was later propagated by Albert H. Ebeling for 34 years.

Eukaryotic Cells in Culture

Eukaryotic cells are much more difficult to culture than most prokaryotic cells.
They require complex culture media and are highly susceptible to contamination and overgrowth by microorganisms such as bacteria, yeast, and fungi.
Some important characteristics of animal cells and their growth in culture can be understood through different animal tissue culture systems.

Types of Animal Tissue Culture

1. Organ Culture

In organ culture, whole embryos, organs, or tissues are removed from the body either by vivisection or shortly after brain death.
The normal physiological functions of the organs or tissues are maintained in this system.
The cells remain fully differentiated in organ culture.
Scale-up is not recommended in organ culture systems.
Organ culture shows slow growth, and fresh explantation is required for every experiment.

2. Tissue Culture

Tissue culture refers to the growth of fragments of excised tissue in a defined culture medium.
Most of the normal functions of the cultured tissue are maintained, even though the original organization of the tissue is lost.
Tissue culture is better for scale-up compared to organ culture, but it is still not ideal.

3. Cell Culture

In cell culture, excised tissues or tissue outgrowths are dispersed, usually by enzymatic methods, into a suspension of individual cells.
These cells may be grown as a monolayer or as a suspension culture.
Cell cultures can be maintained for long periods as specific types of cell lines.
Cell cultures are very suitable for scale-up studies.
Over time, however, cultured cells may lose some of their differentiated characteristics.

Contact Inhibition

Animal cells, unlike plant and microbial cell cultures, show limited growth even when the best nutritive and physical conditions are provided.
The behavior of animal cells in culture depends on the source organ or tissue from which the cells are taken.
In most cases, animal cells grow and divide only for a limited number of generations and then either die or stop growing, even when all appropriate physical and nutritional conditions are available.
Therefore, mortality is associated with all animal cell cultures unless the cells belong to an immortal cell line or a cancer cell line.
Cells derived from tissues such as kidneys, liver, muscles, and embryos grow and spread as a monolayer, filling the surface of the culture container.
When these cells spread over the entire surface and come in contact with the walls of the culture plate or container, they stop further growth, a phenomenon known as contact inhibition.

Behavior of Normal and Cancer Cells in Culture

Cells in culture may exist under completely altered hormonal conditions, and the three-dimensional architecture that was present in the original tissue is totally absent in cell cultures.
Cancer cells behave differently from normal cells.
They lose the phenomenon of contact inhibition and, instead of growing as a monolayer, they pile up on one another to form multilayered cell cultures due to uncontrolled cell division.
These cancer cells are usually round in shape and show continuous cell multiplication when proper nutritional conditions are provided.
Oncologists use these properties of cancer cells, especially their growth pattern, to help confirm the cancerous nature of tumors.

Cell Morphology Categories

Most mammalian cells in culture can be divided into three basic categories based on their morphology.

1. Fibroblastic (Fibroblast-like) Cells

These cells are bipolar or multipolar in shape.
They have an elongated appearance.
They grow attached to a solid substrate or surface.

2. Epithelial-like Cells

These cells are polygonal in shape.
They have more regular and uniform dimensions
They grow attached to a substrate in discrete patches or sheets.

3. Lymphoblast-like Cells

These cells are spherical in shape.
They usually grow in suspension.
They do not attach to a solid surface.

Classification of Animal Cell Culture

Animal cell cultures can be classified into two main types:

Primary Cell Culture
Secondary Cell Culture

1. Primary Cell Culture

Primary cell culture refers to the cell culture that is initially derived directly from the parent tissue before any further culturing in vitro.
The tissues used to establish primary cell cultures are obtained from specific organs such as the kidney or liver.
Examples of primary cell cultures include white blood cells, fibroblasts derived from skin, smooth muscle cells of the heart, neurons, and similar cell types.
These cells may be capable of undergoing one or two divisions in culture and, if provided with suitable conditions, can survive for a limited period.
However, they do not continue to grow indefinitely and eventually undergo senescence and die.
A major drawback of primary cell culture is that it takes a long time to establish a specific type of culture.
Each time, a fresh organ, tissue, or a live organism is required to initiate a new primary culture.

Methods of Obtaining Cell Culture

There are two basic methods used to obtain cell cultures.

1. Explant Culture

In this method, a small piece of tissue is transferred into a glass or plastic culture vessel.
The tissue is bathed in a suitable culture medium.
After a few days, individual cells migrate out from the tissue explant onto the surface of the culture vessel.
These cells then begin to divide and grow.

2. Enzymatic Dissociation

This is the most widely used method because it is faster.
Proteolytic enzymes such as trypsin or collagenase are added to the tissue fragments to dissolve the material that holds the cells together.
This process creates a suspension of single cells.
These cells are then transferred into culture vessels containing fresh culture medium, where they grow and divide.

Cell Culture Systems

The type of cell culture system mainly depends on the source tissue of the organism from which the cells are obtained.
Cells may be grown as suspension cultures, where they float freely in the culture medium, for example, lymphoid cells (white blood cells).
Cells may also be grown as anchored or monolayer cultures, where they adhere to the surface of a glass or treated plastic vessel.
Monolayer cultures are commonly grown in culture-treated dishes, T-flasks, roller bottles, cell stack culture chambers, or multi-well plates.
Suspension cultures are usually grown in spinner flasks, Erlenmeyer flasks, or similar flasks and bottles.

2. Secondary Cell Culture

Primary cell cultures can be maintained and made viable for a long time by subculturing, with or without cell division.
Once a primary cell culture is split or subcultured, it becomes a secondary cell culture.
This process can lead to the formation of a cell line with specific physiological or genetic characteristics.
Under proper physical and nutritional conditions, these cells can divide and grow in vitro for about 50 to 100 generations.
However, they do not divide indefinitely, and over time their physical characteristics may change, after which the cells undergo senescence and die.

Factors Affecting Subculturing Frequency

The frequency of subculturing depends on the type and source of the cells.
Cells derived from differentiated or terminally differentiated tissues divide slowly and therefore require less frequent subculturing.
Cells derived from embryos multiply rapidly and require frequent subculturing to prevent confluency, which can negatively affect cell stability.
Frequent subculturing of slow-growing cultures can result in low cell density and may eventually lead to the loss of cell lines.

Subculturing

1. Subculturing of Suspension Cultures

Subculturing suspension cultures is relatively simple.
It involves diluting cultures with high cell density using fresh nutrient medium, or
Transferring cells from the old culture into fresh medium in sufficient quantity to maintain the desired initial cell density.

2. Subculturing of Anchored (Adherent) Cultures

This process requires a special procedure.
The growth medium is first removed from the culture vessel.
The culture surface is washed several times with fresh medium.
The attached cells are then removed or dissociated from the surface using proteolytic enzymes such as proteases and collagenase.
The detached cells are transferred into fresh culture vessels containing fresh culture medium.
In some cases, cells may also be removed mechanically using a plastic spatula or by gentle pipetting.

Cell Lines and Their Types

A cell line arises from a primary cell culture at the time of the first successful subculture.

Cell lines are broadly classified into two main types:

1. Finite cell lines

2. Immortal (continuous) cell lines

1. Finite Cell Lines (Normal Cell Lines)

Finite cell lines consist of normal cells that undergo a limited number of cell divisions.
Typically, these cells divide up to about 50 generations before they gradually lose vigor and die.
This limited lifespan is mainly due to their inability to synthesize the enzyme telomerase.
Normal cells exhibit the phenomenon of contact inhibition.
Because of contact inhibition and limited division capacity, these cells show restricted growth and eventually undergo senescence and death.

2. Continuous Cell Lines (Immortal Cell Lines)

In some cases, secondary cell cultures may spontaneously undergo transformation, resulting in the formation of cell lines with indefinite growth potential or immortality, such as cancer cell lines.
Such cell lines are known as immortal or continuous cell lines.
Immortal cells continue to grow and divide indefinitely in vitro as long as proper culture conditions and adequate nutrients are provided.
Immortalized cell lines are also referred to as transformed cells, meaning their growth properties have been altered.
HeLa cells are the classic and most widely known example of an immortalized cell line.

Characteristics of Immortal and Cancer Cells

Immortal or transformed cell lines are not necessarily cancerous or tumor cells.
True tumor or cancer cells are capable of forming tumors when introduced into an experimental animal.
In contrast, a transformed or immortalized cell line derived from a normal secondary cell culture does not necessarily form tumors in experimental animals.
Cancer cells in culture produce telomerase, which allows them to divide continuously without losing their division capacity.
Cancer cells do not show contact inhibition.
Once the surface of the culture vessel is covered, these cells continue to divide and pile up into multilayered mounds.

Commonly Used Cell Lines

Human Cell Lines

MCF-7 (breast cancer cell line)
HL-60 (leukemia cell line)
HEK-293 (human embryonic kidney cell line)
HeLa (derived from Henrietta Lacks)

Primate Cell Lines

Vero (African green monkey kidney epithelial cells)
COS-7 (African green monkey kidney cells)

Physical Environment for Culturing Animal Cells

Animal cells are considerably more difficult to culture than most prokaryotic organisms.
They require complex culture media and are highly susceptible to contamination and overgrowth by microorganisms such as bacteria, yeast, and fungi.
Animal cells are extremely sensitive to environmental factors created under artificial in vitro conditions.
The major components of the physical environment required for animal cell culture include temperature, pH, osmolarity, and the gaseous environment.
In addition to supplying essential nutrients and hormonal requirements, the culture medium also plays a protective role by shielding cells from various chemical, physical, and mechanical stresses.

Culture Media

Culture media is a complex mixture containing salts, carbohydrates, vitamins, amino acids, metabolic precursors, growth factors, hormones, and trace elements required for cell survival and growth.
A wide variety of culture media have been formulated for the cultivation of different types of animal cells.
Eagle’s Minimum Essential Medium (EMEM) was one of the first widely used culture media and was formulated by Harry Eagle.
Basal Medium Eagle (BME) was developed specifically for culturing mouse L cells and HeLa cells.
Dulbecco’s Modified Eagle’s Medium (DMEM) contains approximately twice the concentration of amino acids and four times the amount of vitamins compared to EMEM.
Iscove’s Modified Dulbecco’s Medium (IMDM) was formulated for the growth of lymphocytes and hybridomas.
Hybri-Care Medium is used for the propagation of hybridomas and other fastidious cell lines.
McCoy’s 5A and RPMI-1640 are commonly used media that support the growth of primary cell cultures.

Components of Basal Media

1. Carbon Sources

Carbohydrates are mainly supplied in the form of glucose.
In some cases, glucose is replaced with galactose to reduce lactic acid accumulation in the medium.
Other important carbon sources include amino acids, particularly L-glutamine, and pyruvate.

2. Buffers

The optimal pH range of 7.2 to 7.4 is maintained by adding one or more buffering systems to the medium.
CO₂/sodium bicarbonate–buffered media require a controlled CO₂ atmosphere.
HEPES is a strong chemical buffer effective in the pH range of 7.2 to 7.6 and does not require a CO₂ atmosphere.
Sera can also contribute to buffering the complete culture medium.

3. Inorganic Salts

Inorganic salts help maintain osmolarity of the culture medium.
They regulate membrane potential through ions such as Na⁺, K⁺, and Ca²⁺.
These ions are also required for cell attachment and act as cofactors for various enzymes.

4. pH Indicator

Phenol red is commonly used as a pH indicator in tissue culture media.
It allows colorimetric monitoring of pH changes during cell growth.
As cells metabolize nutrients and release metabolites, the pH of the medium changes, resulting in a color change.
At low pH, phenol red turns the medium yellow, while at high pH it turns purple.
For most tissue culture applications at pH 7.4, the medium should appear bright red.

5. Keto Acids (Oxaloacetate and Pyruvate)

Keto acids such as oxaloacetate and pyruvate are intermediates of glycolysis and the Krebs cycle.
They are added to culture media as energy sources and as carbon skeletons for anabolic processes.
Cellular metabolism of pyruvate produces carbon dioxide, which diffuses into the atmosphere and forms bicarbonate in the medium.
These compounds help maintain maximum cellular metabolic activity.

6. Vitamins

Vitamins act as precursors for numerous enzyme cofactors.
Common vitamins present in basal media include riboflavin, thiamine, and biotin.

7. Trace Elements

Trace elements required in small amounts include zinc, copper, selenium, and intermediates of the tricarboxylic acid cycle.

8. Supplements

Some cell lines require complete growth media supplemented with additional components not present in the base medium or serum.
These supplements include hormones, growth factors, and signaling molecules that support proliferation and maintain normal cellular metabolism.

a) L-Glutamine

L-Glutamine is an essential amino acid for cultured cells.
It is required for protein synthesis, energy production, and nucleic acid metabolism.
L-Glutamine is unstable in liquid media and is therefore added separately as a supplement.

b) Non-Essential Amino Acids (NEAA)

Most media formulations contain the ten essential amino acids along with non-essential amino acids such as cysteine, glutamine, and tyrosine.
Additional non-essential amino acids include alanine, asparagine, aspartic acid, glycine, glutamic acid, proline, and serine.
The presence of non-essential amino acids reduces the metabolic burden on cells and enhances cellular proliferation.

c) Growth Factors and Hormones

Growth factors and hormones are required for optimal cell growth and maintenance of differentiation.
For example, insulin stimulates glucose transport and utilization and enhances amino acid uptake.

d) Antibiotics and Antimycotics

Antibiotics and antimycotic agents are added to culture media to prevent bacterial and fungal contamination.
Routine use of antibiotics or antimycotics is not recommended unless specifically required.
Common examples include penicillin, streptomycin, gentamicin, and amphotericin B.

Osmolarity and Temperature of Culture Medium

Osmolarity

The osmolarity of a culture medium is determined by the concentration of salts and other water-soluble substances such as glucose, amino acids, and related components present in the medium.
The osmolarity of the medium can be accurately monitored using an osmometer.
The recommended osmolarity for most animal cell culture media is approximately 300 mOsm.
Any alteration in the osmolarity of the culture medium can significantly affect cell growth and cellular metabolism.
Extreme changes in osmolarity may lead to cellular damage and can ultimately result in cell death.

Temperature

Temperature is an important physical factor that directly influences the physiology and metabolic activity of living cells.
The optimal temperature for the in vitro cultivation of most animal cells is 37°C, which closely resembles normal physiological body temperature.

Types of Culture Media

Culture media can be classified into two main types based on their source and composition.

1. Natural Media

Media prepared from natural sources are referred to as natural media.
These media contain complex biological components that support cell growth.
Examples of natural media include blood serum, various body fluids such as amniotic fluid, tissue extracts, and embryo extracts.

2. Synthetic Media

Media prepared from defined salt solutions and other components mixed in predetermined proportions are known as synthetic media.
Synthetic media provide more controlled and reproducible conditions for cell culture.

Major Equipment Used in Animal Cell Culture Laboratory

1. Laminar Air Flow (LAF) Chamber

A laminar air flow chamber provides a sterile, microbe-free working environment.
It works by continuously passing filtered air over the workbench surface.
All aseptic procedures such as handling cells and tissues, implanting cells or tissues into culture media, and performing culture-related experiments are carried out inside this chamber.

2. Carbon Dioxide (CO₂) Incubator

A carbon dioxide incubator is specifically designed for incubating animal cell cultures.
It maintains a constant temperature, sterility, humidity, and an optimal concentration of carbon dioxide.
The CO₂ atmosphere helps maintain the pH of the culture medium.
It also closely mimics the physiological environmental conditions required for living cells.

3. Inverted Microscope

Inverted microscopes are commonly used in animal cell culture laboratories.
They allow observation of cells in situ, meaning the cells can be viewed while still inside the culture vessel.
This enables monitoring of cell growth without disturbing the culture conditions.
Inverted microscopes are especially useful for observing adherent cells growing as a monolayer at the bottom of culture plates or flasks.

4. Centrifuges

A low-speed, tabletop refrigerated centrifuge is recommended for animal cell culture work.
It is used to isolate cells from suspension cultures.
It also helps in separating cells from the liquid phase during washing or harvesting procedures.

Other Essential Instruments

Autoclave – used for sterilizing non–heat-labile liquids and materials.
Sterilizing oven – used for sterilizing glassware and metallic instruments.
Refrigerators and freezers – used for storing culture media, reagents, and frozen serum.
Cryostorage systems – used for long-term storage of cell stocks at very low temperatures.
Fluid-handling systems – include pipettors, micropipettes, and multichannel pipettors for accurate liquid handling.

These instruments together ensure proper maintenance, handling, and long-term preservation of animal cell cultures under sterile and controlled conditions.

Equipments Required for Animal-Cell Culture

Cryopreservation of Cell Culture

During prolonged subculturing, several factors can affect the properties and stability of cultured cells.
When cells need to be stored for long periods, freezing and storage is the preferred method.
Cryopreservation refers to the storage of cells and tissues at extremely low temperatures, typically at liquid nitrogen temperatures ranging from –180°C to –196°C.
Freezing cells at liquid nitrogen temperature allows them to be preserved in an intact state without significant changes for long durations.

Cell Damage During Freezing

Freezing of cells and tissues can cause serious damage, mainly due to the formation of intracellular ice crystals.
Additional damage may result from severe dehydration and denaturation of cellular proteins.
These harmful effects can be minimized by using preservative agents and applying slow cooling methods.
Slow cooling allows water to move out of the cells before freezing, reducing intracellular ice formation.

Low-Temperature Freezing Strategy

Freezing at very low temperatures, such as –180°C, retards the formation and growth of ice crystals inside the cells.
One method involves freezing cells at –20°C for one hour, followed by transfer to –70°C overnight.
On the following day, the cells are transferred to liquid nitrogen for long-term storage.

Use of Cryoprotective Agents

Another effective method of cryopreservation involves the use of cryoprotective agents.
Cryoprotective agents prevent the formation and growth of ice crystals within cells.
Certain media components such as sugars, serum, and some solvents can function as cryoprotectants.
The most commonly used cryoprotective agents are dimethyl sulfoxide (DMSO) and glycerol.
These antifreeze agents bind water molecules strongly, preventing them from freezing at 0°C.
The absence of ice crystal formation helps maintain membrane integrity and overall cell viability during storage.

Procedure of Cryopreservation of Cells

Remove the growth medium from the culture vessel and wash the cells with phosphate-buffered saline (PBS), then remove the PBS completely.
Dislodge the adherent cells using trypsin.
Dilute the detached cells with fresh growth medium.
Transfer the cell suspension into a 15 mL conical tube and centrifuge at 200 g for 5 minutes at room temperature (RT), then remove the growth medium by aspiration.
Resuspend the cell pellet in 1–2 mL of freezing medium.
Transfer the cell suspension into cryovials.
Incubate the cryovials at –80°C overnight.
On the next day, transfer the cryovials into liquid nitrogen for long-term storage.

Advantages of Cryopreservation

Cryopreservation helps prevent genotypic drift caused by genetic instability.
It prevents cellular senescence and cell death.
It reduces the risk of transformation of cells into cancerous or immortal cell lines.
It minimizes phenotypic instability in cultured cells.
Cryopreservation can help prevent microbial contamination.
It reduces the chances of cross-contamination with other cell lines.
This technique saves time and laboratory resources when cells are not in immediate use.
It allows easy storage and distribution of cell lines to other users.

Working with Cryopreserved Cells (Thawing)

Cryopreserved cells must be brought back to room temperature or incubator temperature before use in culture experiments.
This process is known as thawing and must be performed carefully.
The cryovial removed from liquid nitrogen should be immediately placed in a 37°C water bath.
Cells should be thawed rapidly to prevent ice crystal formation.
Centrifuge the vial for 10 minutes at 1000 rpm at room temperature, wipe the top of the vial with 70% ethanol, and discard the supernatant.
Resuspend the cell pellet in 1 mL of complete medium with 20% PBS and transfer it to a properly labeled culture plate containing the appropriate amount of medium.
Examine the cultures after 24 hours to ensure that the cells have attached to the surface.
Change the culture medium as its color changes.

Cell Counting

Cell counting is necessary to establish or monitor growth rates and to set up new cultures with known cell numbers.
Hemocytometers are commonly used to estimate cell number and assess cell viability.
A hemocytometer is a thick glass slide containing two counting chambers, one on each side.
Each counting chamber has a mirrored surface with a 3 × 3 mm grid divided into 9 counting squares.
Raised sides of the chamber hold a coverslip exactly 0.1 mm above the chamber floor.
Each of the 9 counting squares corresponds to a volume of 0.0001 mL.
Hemocytometers are excellent for determining cell viability but are less precise for determining total cell number due to the limited number of cells counted.
Automated cell counters, such as a Cellometer, provide more reliable and reproducible data.
Cellometers also allow visualization of cell morphology for visual confirmation after counting.
These systems save sample images along with results securely on the computer and can automatically save data on a network for improved data protection and convenience.

Cell Viability

Cell viability assays are used to measure the number of living (viable) cells present in a cell population.
When the number of viable cells is considered along with the total cell count, it provides an accurate indication of the overall health and condition of a cell culture.
The most common and rapid viability assays are based on the integrity of the cell membrane as an indicator of cell viability.
Dyes such as trypan blue and erythrosin B are excluded by viable cells with intact membranes but are taken up and retained by non-viable (dead) cells with damaged membranes.
When using trypan blue, cells must be incubated with the dye for 2–5 minutes before counting.
Erythrosin B does not require an incubation period before use.
Erythrosin B provides more accurate results with fewer false positives and false negatives.
Erythrosin B produces a clearer background and does not bind serum proteins as strongly as trypan blue, making stained cells easier to identify.
Trypan blue is toxic and is considered a potential carcinogen.

Procedure for Cell Viability Assay

Mix the cell suspension in a 1:1 ratio with either 0.1% erythrosin B solution in PBS or 0.4% trypan blue solution in PBS.
Load the erythrosin B–treated cells directly into a clean, dry hemocytometer.
For trypan blue–treated cells, incubate the mixture for 2–5 minutes before loading into the hemocytometer.
Non-viable cells appear red when stained with erythrosin B or dark blue when stained with trypan blue.
Viable cells remain unstained.

Calculation of Cell Viability

Cell viability is calculated by dividing the number of unstained (viable) cells by the total number of cells and expressing the result as a percentage.
Percentage of viable cells = Number of unstained cells × 100 / Total number of cells

Confluency

Confluency is a term commonly used in cell culture biology to estimate the proportion of the surface area of a culture vessel that is covered by adherent cells.
For example, 50% confluency indicates that approximately half of the surface is covered by cells, leaving space for further growth.
100% confluency means the entire surface is covered by cells, with no remaining space for additional monolayer growth.
Cells are usually passaged (subcultured) before they reach full confluency in order to maintain healthy proliferation and normal cellular phenotype.

Applications of Animal Cell Culture Technology

Animal cell culture technology is widely used for the industrial production of therapeutic proteins, hormones, antibodies, and vaccines.

Major Applications

1. Vaccine production

2. Monoclonal antibody production

3. Enzyme and hormone production

4. In vitro tissue and organ culture

5. Viral cultivation

1. Enzymes and Hormones Production

CHO Cell Lines

Chinese Hamster Ovary (CHO) cells are the most widely used mammalian cell lines for commercial-scale production of therapeutic proteins using recombinant DNA technology.

a. Tissue Plasminogen Activator (t-PA)

An anti-clot protease used to dissolve blood clots.
Used in heart attacks and other clot-related conditions.
Produced by genetically engineered mammalian cells.
One of the first mammalian cell culture products to reach the market.
Acts by converting plasminogen into plasmin, which dissolves clots.

b. Factor VIII and Factor IX

Essential blood clotting proteins.
Absence or malfunction causes hemophilia:

Hemophilia A → Factor VIII deficiency
Hemophilia B → Factor IX deficiency

Produced using recombinant DNA technology in mammalian cell cultures and are commercially available.

c. Erythropoietin (EPO)

A hormone-like growth factor responsible for red blood cell production.
Released during hypoxia or anemia.
Used in treatment of anemia associated with:

Surgery
Chemotherapy
AIDS
Cancer
Renal failure

Recombinant human EPO (r-HuEPO) is produced using mammalian cell culture.
Preferred over blood transfusion due to reduced risk and no donor requirement.

2. Monoclonal Antibody Production

Polyclonal vs Monoclonal Antibodies

Polyclonal antibodies: Produced by different B-cells; heterogeneous.
Monoclonal antibodies: Produced by a single B-cell clone; identical and specific.

Hybridoma Technology

Antigen is injected into a mouse.
Antibody-producing spleen cells are isolated.
These cells are fused with immortal cancerous immune cells.
The fused cell is called a hybridoma.
Desired hybridomas are selected, cloned, and cultured to produce monoclonal antibodies continuously.

3. In Vitro Skin and Organ Tissue Culture

Stem Cells

Primitive cells capable of differentiating into various cell types.
Can be isolated from:

Blood
Brain
Muscle tissue
Early human embryos

Advantages of Embryonic Stem Cells

Pluripotent “master cells”
Can develop into almost any tissue type

Applications

In vitro growth of:

Skin cells
Cardiac tissues
Blood and hematopoietic cells

Cultured skin sheets are used for burn treatment and grafting.

Scale-Up of Animal Cell Culture

Definition

Scale-up refers to the conversion of laboratory-scale cell culture into an industrial-scale process, using principles of biochemical engineering.

Challenges in Scale-Up

Animal cells are large and fragile.
Many animal cells are anchor-dependent.
Complex and expensive nutritional requirements.
Media are prone to contamination.
Low cell density and low product yield.
Costly downstream processing for purification.

Scale-Up Techniques

Fermentors used despite limitations.
Cell lines like BHK-21 grown in fermentors for vaccine production.
Roller bottles and microcarriers for anchor-dependent cultures.
Spinner flasks for suspension cultures.

Animal Cell Culture: Definition, Media, Growth, Techniques, Applications & Scale-Up - Biotechnology Lecture